Polymethine compounds and their use as fluorescent labels

ABSTRACT

The present disclosure relates to new compounds and their use as fluorescent labels. The compounds may be used as fluorescent labels for nucleotides in nucleic acid sequencing applications.

The present application is a U.S. national phase under 35 U.S.C. § 371of International Patent Application No. PCT/GB2016/052986, filed Sep.26, 2016, which claims the benefit of priority to United Kingdom (GB)Appl. No. 1516987.3, filed Sep. 25, 2015, which is incorporated byreference.

The present disclosure relates to new polymethine compounds and theiruse as fluorescent markers. In particular the compounds may be used asfluorescent labels for nucleotides in nucleic acid sequencingapplications.

BACKGROUND

Several publications and patent documents are referenced in thisapplication in order to more fully describe the state of the art towhich this disclosure pertains. The disclosure of each of thesepublications and documents is incorporated by reference herein.

Non-radioactive detection of nucleic acids utilizing fluorescent labelsis an important technology in molecular biology. Many proceduresemployed in recombinant DNA technology previously relied heavily on theuse of nucleotides or polynucleotides radioactively labelled with, forexample ³²P. Radioactive compounds permit sensitive detection of nucleicacids and other molecules of interest. However, there are seriouslimitations in the use of radioactive isotopes such as their expense,limited shelf life and more importantly safety considerations.Eliminating the need for radioactive labels enhances safety whilstreducing the environmental impact and costs associated with, forexample, reagent disposal. Methods amenable to non-radioactivefluorescent detection include by way of non-limiting example, automatedDNA sequencing, hybridization methods, real-time detection ofpolymerase-chain-reaction products and immunoassays.

For many applications it is desirable to employ multiple spectrallydistinguishable fluorescent labels in order to achieve independentdetection of a plurality of spatially overlapping analytes. In suchmultiplex methods the number of reaction vessels may be reduced,simplifying experimental protocols and facilitating the production ofapplication-specific reagent kits. In multi-colour automated DNAsequencing for example, multiplex fluorescent detection allows for theanalysis of multiple nucleotide bases in a single electrophoresis lane,thereby increasing throughput over single-colour methods and reducinguncertainties associated with inter-lane electrophoretic mobilityvariations.

However, multiplex fluorescent detection can be problematic and thereare a number of important factors which constrain selection offluorescent labels. First, it may be difficult to find dye compoundswhose emission spectra are suitably spectrally resolved in a givenapplication. In addition when several fluorescent dyes are usedtogether, to generate fluorescence signals in distinguishable spectralregions by simultaneous excitation may be difficult because theabsorption bands of the dyes which could be useable for this are usuallywidely separated, so it is difficult to achieve more or less equalfluorescence excitation efficiency even for two dyes. Many excitationmethods use high power light sources like lasers and therefore the dyemust have sufficient photo-stability to withstand such excitation. Afinal consideration of particular importance in molecular biologymethods is the extent to which the fluorescent dyes must be compatiblewith the reagent chemistries used such as for example DNA synthesissolvents and reagents, buffers, polymerase enzymes and ligase enzymes.

As sequencing technology advances a need has developed for furtherfluorescent dye compounds, their nucleic acid conjugates and dye setswhich satisfy all of the above constraints and which are amenableparticularly to high throughput molecular methods such as solid phasesequencing and the like.

Fluorescent dye molecules with improved fluorescence properties such asfluorescence intensity, shape and wavelength maximum of fluorescenceband can improve the speed and accuracy of nucleic acid sequencing.Strong fluorescence signal is especially important when measurements aremade in water-based biological buffers and at higher temperature as thefluorescence intensity of most dyes is significantly lower at suchconditions. Moreover, the nature of the base to which a dye is attachedalso affects the fluorescence maximum, fluorescence intensity and othersspectral dye properties. The sequence specific interactions between thenucleobases and the fluorescent dyes can be tailored by specific designof the fluorescent dyes. Optimisation of the structure of thefluorescent dyes can improve the efficiency of nucleotide incorporation,reduce the level of sequencing errors and decrease the usage of reagentsin, and therefore the costs of, nucleic acid sequencing.

Described herein are improved polymethine constructs and their use asbio-molecule labels, particularly as labels for nucleotides used innucleic acid sequencing. Particular improvements can be seen in theefficiency of labelled nucleotide incorporation and length and accuracyof sequencing read obtainable using the new fluorescent constructs. Themolecules described below are particularly advantageous in situationswhere high power excitation sources are used. High power excitation canresult in a degree of power saturation, where the number of photonsemitted by the labels ceases to be linear with an increase in excitationpower. Power saturation can be caused by molecular quenching effectsrelating to the structure of the labels, or can be caused by labelshaving a longer than desired fluorescence lifetime, whereby the labelsspend a longer period than desired in the excited state before emittinga photon. Where multiple different labels are used in parallel, havingone of the labels reaching power saturation when other labels continueto get brighter makes differentiation of the labels more difficult.Labels described herein are particularly advantageous in reaching powersaturation at a higher level than corresponding prior art labellingcompounds.

SUMMARY

According to a first aspect this disclosure provides compounds of theformula (I) or mesomeric forms thereof:

wherein mCat+ or mAn− is an organic or inorganic positively/negativelycharged counterion and

m is an integer 0-3;

p is an integer 1-2;

q is an integer 1-5;

alk is a chain of 1-5 carbon atoms optionally containing one or moredouble or triple bonds;

Y is S, O or CH₂;

Z is OH;

n is an integer 0-3;

X is OH or O⁻ or an amide or ester conjugate thereof;

each of Ra₁ and Ra₂ is independently H, SO₃ ⁻, sulfonamide, halogen, ora further ring fused to an adjacent carbon atom;

each of Rc₁ and Rc₂ is independently alkyl or substituted alkyl.

P can be one; in which case the compounds are of the formula (Ia) ormesomeric forms thereof:

wherein mCat+ or mAn− is an organic or inorganic positively/negativelycharged counterion and

m is an integer 0-3;

q is an integer 1-5;

alk is a chain of 1-5 carbon atoms optionally containing one or moredouble or triple bonds;

Y is S, O or CH₂;

Z is OH;

n is an integer 0-3;

X is OH or O⁻ or an amide or ester conjugate thereof;

each of Ra₁ and Ra₂ is independently H, SO₃ ⁻, sulfonamide, halogen, ora further ring fused to an adjacent carbon atom;

each of Rc₁ and Rc₂ is independently alkyl or substituted alkyl.

P can be two; in which case the compounds are of the formula (Ib) ormesomeric forms thereof:

wherein mCat+ or mAn− is an organic or inorganic positively/negativelycharged counterion and

m is an integer 0-3;

q is an integer 1-5;

alk is a chain of 1-5 carbon atoms optionally containing one or moredouble or triple bonds;

Y is S, O or CH₂;

Z is OH;

n is an integer 0-3;

X is OH or O⁻ or an amide or ester conjugate thereof;

each of Ra₁ and Ra₂ is independently H, SO₃ ⁻, sulfonamide, halogen, ora further ring fused to an adjacent carbon atom;

each of Rc₁ and Rc₂ is independently alkyl or substituted alkyl.

In certain examples where Ra₁ or Ra₂ is H; either Rc₁ or Rc₂ can be analkyl sulfonic acid group. The compounds require one or more sulfonicacid substituents, such that at least one of Ra₁ or Ra₂ is SO₃ ⁻, or Ra₁or Ra₂ is a further ring fused to an adjacent carbon atom, the furtherring having an SO₃ ⁻, or Rc₁ or Rc₂ is an alkyl sulfonic acid group.

In certain examples where p is 2, Rc₁ or Rc₂ is an alkyl sulfonic acidgroup.

In certain examples r equals 3.

In certain examples n is 1. The OH group may be at the 4-position on thering. In certain examples n is 0 and the phenyl ring is unsubstituted.In certain examples where n is 0, Y is S or O (and not CH₂).

In another embodiment the compounds of the present disclosure can beconjugated with a variety of substrate moieties such as, for example,nucleosides, nucleotides, polynucleotides, polypeptides, carbohydrates,ligands, particles, cells, semi-solid surfaces (e.g. gels) and solidsurfaces. The conjugation can be carried out via carboxyl group C(═O)—X,which can be turned into an amide or ester.

According to a further aspect of the disclosure therefore, there areprovided dye compounds comprising linker groups to enable, for example,covalent attachment to such substrate moieties.

According to a further aspect the disclosure provides a nucleoside ornucleotide compound defined by the formula: N-L-Dye, wherein N is anucleotide, L is an optional linker moiety and Dye is a fluorescentcompound according to the present disclosure.

In a further aspect the disclosure provides methods of sequencing usingthe dye compounds of the present disclosure.

According to a further aspect the disclosure also provides kitscomprising dye compounds (free or in conjugate form) which may be usedin various immunological assays, oligonucleotide and nucleic acidlabelling and for DNA sequencing by synthesis. In yet another aspect thedisclosure provides kits comprising dye ‘sets’ particularly suited tocycles of sequencing by synthesis on an automated instrument platform.

A further aspect of the disclosure is the chemical preparation ofcompounds of the disclosure.

DETAILED DESCRIPTION

This disclosure provides novel compounds particularly suitable formethods of fluorescence detection and sequencing by synthesis. Compoundshaving an indole N-phenyl moiety are advantageous in fluorescenceintensity, photostability compared to N-alkyl analogues and thereforeimprove certain nucleic acid sequencing applications.

According to a first aspect the disclosure provides compounds of theformula (I) or mesomeric forms thereof:

wherein mCat+ or mAn− is an organic or inorganic positively/negativelycharged counterion and

m is an integer 0-3;

p is an integer 1-2;

q is an integer 1-5;

alk is a chain of 1-5 carbon atoms optionally containing one or moredouble or triple bonds;

Y is S, O or CH₂;

Z is OH;

n is an integer 0-3;

X is OH or O⁻ or an amide or ester conjugate thereof;

each of Ra₁ and Ra₂ is independently H, SO₃ ⁻, sulfonamide, halogen, ora further ring fused to an adjacent carbon atom;

each of Rc₁ and Rc₂ is independently alkyl or substituted alkyl.

The disclosure provides a compound of the formula (I) or mesomeric formsthereof:

wherein mCat+ or mAn− is an organic or inorganic positively/negativelycharged counterion and

m is an integer 0-3;

p is an integer 1-2;

q is an integer 1-5;

alk is a chain of 1-5 carbon atoms optionally containing one or moredouble or triple bonds;

Y is S, O or CH₂;

Z is OH;

n is an integer 0-3;

X is OH or O⁻ or an amide or ester conjugate thereof;

each of Ra₁ and Ra₂ is independently H, SO₃ ⁻, sulfonamide, halogen, ora further ring fused to an adjacent carbon atom; and

each of Rc₁ and Rc₂ is independently alkyl or substituted alkyl, whereinat least one of Ra₁ or Ra_(z) is SO₃ ⁻, or Ra₁ or Ra₂ is a further ringfused to an adjacent carbon atom, the further ring having an SO₃ ⁻, orRc₁ or Rc₂ is an alkyl sulfonic acid group.

The disclosure provides a compound of the formula (I) or mesomeric formsthereof:

wherein mCat+ or mAn− is an organic or inorganic positively/negativelycharged counterion and

m is an integer 0-3;

p is an integer 1-2;

q is an integer 1-5;

alk is a chain of 1-5 carbon atoms optionally containing one or moredouble or triple bonds;

Y is S, O or CH₂;

Z is OH;

n is an integer 0-3;

X is OH or O⁻ or an amide or ester conjugate thereof; each of Ra₁ andRa₂ is independently H, SO₃ ⁻, sulfonamide, halogen, or a further ringfused to an adjacent carbon atom; and

each of Rc₁ and Rc₂ is independently alkyl or substituted alkyl, whereinwhen n is 0, Y is S or O.

The disclosure provides a compound of the formula (I) or mesomeric formsthereof:

wherein mCat+ or mAn− is an organic or inorganic positively/negativelycharged counterion and

m is an integer 0-3;

p is an integer 1-2;

q is an integer 1-5;

alk is a chain of 1-5 carbon atoms optionally containing one or moredouble or triple bonds;

Y is S, O or CH₂;

Z is OH;

n is an integer 0-3;

X is OH or O⁻ or an amide or ester conjugate thereof;

each of Ra₁ and Ra₂ is independently H, SO₃ ⁻, sulfonamide, halogen, ora further ring fused to an adjacent carbon atom; and

each of Rc₁ and Rc₂ is independently alkyl or substituted alkyl, whereinat least one of Ra₁ or Ra_(z) is SO₃ ⁻, or Ra₁ or Ra₂ is a further ringfused to an adjacent carbon atom, the further ring having an SO₃ ⁻, orRc₁ or Rc₂ is an alkyl sulfonic acid group and wherein when n is 0, Y isS or O.

The molecules may contain one or more sulphonamide or SO₃ ⁻ moieties atposition Ra. Ra₁ and/or Ra_(z) may be SO₃ ⁻ or sulphonamide. The otherRa (Ra₁ or Ra₂) can be independently H, SO₃ ⁻, sulphonamide, halogen, ora further ring fused to an adjacent carbon atom. Ra₁ or Ra₂ can be H.Ra₁ or Ra₂ can be SO₃ ⁻. Ra₁ can be different to Ra₂, for example thestructure can have a single sulfonamide group at Ra₁, and H as Ra₂. Ra₁and Ra₂ can both be sulphonamide. The sulphonamide can be SO₂NH₂ orSO₂NHR, where R is an alkyl, substituted alkyl, aryl or substituted arylgroup. Where neither Ra₁ or Ra₂ is a SO₃ ⁻ or a further ring fused to anadjacent carbon atom, then Rc₁ or Rc₂ must be an alkyl sulfonic acidgroup.

Ra₁ or Ra₂ can be a further aliphatic, aromatic or heterocyclic ringfused to adjacent carbons of the indole ring. For example, in such caseswhen an aromatic ring is fused the dyes end group can represent astructure of type

where Rd can be H, alkyl, substituted alkyl, aryl, substituted aryl,halogen, carboxy, sulphonamide, or sulfonic acid.

Thus the dyes of the disclosure can be described by Formula (1C) or (ID)or mesomeric forms thereof:

wherein mCat+ or mAn− is an organic or inorganic positively/negativelycharged counterion and

m is an integer 0-3;

p is an integer 1-2;

q is an integer 1-5;

alk is a chain of 1-5 carbon atoms optionally containing one or moredouble or triple bonds;

Y is S, O or CH₂;

Z is OH;

n is an integer 0-3;

X is OH or O⁻ or an amide or ester conjugate thereof;

each of Ra₁ and Ra₂ is independently H, SO₃ ⁻, sulfonamide, halogen, ora further ring fused to an adjacent carbon atom;

each of Rc₁ and Rc₂ is independently alkyl or substituted alkyl; and

Rd is H, alkyl, substituted alkyl, aryl, substituted aryl, halogen,carboxy, sulphonamide, or sulfonic acid.

Thus the dyes of the disclosure can be described by Formula (1C) or (ID)or mesomeric forms thereof:

wherein mCat+ or mAn− is an organic or inorganic positively/negativelycharged counterion and

m is an integer 0-3;

p is an integer 1-2;

q is an integer 1-5;

alk is a chain of 1-5 carbon atoms optionally containing one or moredouble or triple bonds;

Y is S, O or CH₂;

Z is OH;

n is an integer 0-3;

X is OH or O⁻ or an amide or ester conjugate thereof;

each of Ra₁ and Ra₂ is independently H, SO₃ ⁻, sulfonamide, halogen, ora further ring fused to an adjacent carbon atom;

each of Rc₁ and Rc₂ is independently alkyl or substituted alkyl; and

Rd is H, alkyl, substituted alkyl, aryl, substituted aryl, halogen,carboxy, sulphonamide, or sulfonic acid, wherein at least one of Ra₁ orRa₂ is SO₃ ⁻, or Rd is SO₃ ⁻, or Rc₁ or Rc₂ is an alkyl sulfonic acidgroup.

Thus the dyes of the disclosure can be described by Formula (1C) or (ID)or mesomeric forms thereof:

wherein mCat+ or mAn− is an organic or inorganic positively/negativelycharged counterion and

m is an integer 0-3;

p is an integer 1-2;

q is an integer 1-5;

alk is a chain of 1-5 carbon atoms optionally containing one or moredouble or triple bonds;

Y is S, O or CH₂;

Z is OH;

n is an integer 0-3;

X is OH or O⁻ or an amide or ester conjugate thereof;

each of Ra₁ and Ra₂ is independently H, SO₃ ⁻, sulfonamide, halogen, ora further ring fused to an adjacent carbon atom;

each of Rc₁ and Rc₂ is independently alkyl or substituted alkyl; and

Rd is H, alkyl, substituted alkyl, aryl, substituted aryl, halogen,carboxy, sulphonamide, or sulfonic acid, wherein

when n is 0, Y is S or O.

Thus the dyes of the disclosure can be described by Formula (1C) or (ID)or mesomeric forms thereof:

wherein mCat+ or mAn− is an organic or inorganic positively/negativelycharged counterion and

m is an integer 0-3;

p is an integer 1-2;

q is an integer 1-5;

alk is a chain of 1-5 carbon atoms optionally containing one or moredouble or triple bonds;

Y is S, O or CH₂;

Z is OH;

n is an integer 0-3;

X is OH or O⁻ or an amide or ester conjugate thereof;

each of Ra₁ and Ra₂ is independently H, SO₃ ⁻, sulfonamide, halogen, ora further ring fused to an adjacent carbon atom;

each of Rc₁ and Rc₂ is independently alkyl or substituted alkyl; and

Rd is H, alkyl, substituted alkyl, aryl, substituted aryl, halogen,carboxy, sulphonamide, or sulfonic acid, wherein at least one of Ra₁ orRa₂ is SO₃ ⁻, or Rd is SO₃ ⁻, or Rc₁ or Rc₂ is an alkyl sulfonic acidgroup and wherein when n is 0, Y is S or O.

In formula (IC) or (ID) the additional rings fused to adjacent carbonatoms of the indole ring may be optionally substituted, for example withsulfonic acid or sulphonamide.

The C(═O)—X carboxy group or its derivatives is attached to the indolenitrogen atom by an alkyl chain of length q, where q is 1-5 carbon orhetero-atoms. The chain may be (CH₂)q where q is 1-5. The group may be(CH₂)₅COOH.

The molecules can contain one or more alkyl-sulfonate moieties atposition Rc. Either Rc₁ and/or Rc₂ may be alkyl-SO₃ ⁻. The other Rc (Rc₁or Rc₂) can be independently alkyl or substituted alkyl. Rc₁ and Rc₂ maybe independently methyl, ethyl, propyl, butyl, pentyl, hexyl or(CH₂)_(t)SO₃H, where t is 1-6. t may be 1-4. t may be 4. Rc₁ and Rc₂ maybe a substituted alkyl group. Rc₁ and Rc₂ may contain a COOH or —SO₃Hmoiety or their ester or amide derivatives.

In certain embodiments, when one of Ra₁ or Ra₂ is SO₃ ⁻, and the otherof Ra₁ or Ra₂ is H or SO₃ ⁻, either Rc₁ or Rc₂ can also be an alkylsulfonic acid group.

The COOH group shown as C(═O)—X can act as a linking moiety for furtherattachment or is linked to a further molecule.

Once conjugation has occurred, the COOH or COO⁻ is converted into anamide or ester.

Examples of compounds include structures according to formula (II) or(IIa) or mesomeric forms thereof:

wherein mCat+ or mAn− is an organic or inorganic positively/negativelycharged counterion and

m is an integer 0-3;

p is an integer 1-2;

q is an integer 1-5;

alk is a chain of 1-5 carbon atoms optionally containing one or moredouble or triple bonds;

Y is S, O or CH₂;

Z is OH;

n is an integer 0-3;

X is OH or O⁻ or an amide or ester conjugate thereof; each of Ra₁ andRa₂ is independently H, SO₃ ⁻, sulfonamide, halogen, or a further ringfused to an adjacent carbon atom; and

each of Rc₁ and Rc₂ is independently alkyl or substituted alkyl.

Examples of compounds include structures according to formula (II) or(IIa) or mesomeric forms thereof:

wherein mCat+ or mAn− is an organic or inorganic positively/negativelycharged counterion and

m is an integer 0-3;

p is an integer 1-2;

q is an integer 1-5;

alk is a chain of 1-5 carbon atoms optionally containing one or moredouble or triple bonds;

Y is S, O or CH₂;

Z is OH;

n is an integer 0-3;

X is OH or O⁻ or an amide or ester conjugate thereof;

each of Ra₁ and Ra₂ is independently H, SO₃ ⁻, sulfonamide, halogen, ora further ring fused to an adjacent carbon atom; and

each of Rc₁ and Rc₂ is independently alkyl or substituted alkyl, whereinwhen n is 0, Y is S or O.

Further examples of compounds include structures according to formula(IIIa) or (IIIb):

wherein mCat+ or mAn− is an organic or inorganic positively/negativelycharged counterion and

m is an integer 0-3;

p is an integer 1-2;

q is an integer 1-5;

alk is a chain of 1-5 carbon atoms optionally containing one or moredouble or triple bonds;

t is an integer 1-6;

Y is S, O or CH₂;

Z is OH;

n is an integer 0-3;

X is OH or O⁻ or an amide or ester conjugate thereof;

each of Ra₁ and Ra₂ is independently H, SO₃ ⁻, sulfonamide, halogen, ora further ring fused to an adjacent carbon atom; and

each of Rc₁ and Rc₂ is independently alkyl or substituted alkyl.

Further examples of compounds include structures according to formula(IIIa) or (IIIb):

wherein mCat+ or mAn− is an organic or inorganic positively/negativelycharged counterion and

m is an integer 0-3;

p is an integer 1-2;

q is an integer 1-5;

alk is a chain of 1-5 carbon atoms optionally containing one or moredouble or triple bonds;

t is an integer 1-6;

Y is S, O or CH₂;

Z is OH;

n is an integer 0-3;

X is OH or O⁻ or an amide or ester conjugate thereof; each of Ra₁ andRa₂ is independently H, SO₃ ⁻, sulfonamide, halogen, or a further ringfused to an adjacent carbon atom; and

each of Rc₁ and Rc₂ is independently alkyl or substituted alkyl, whereinwhen n is 0, Y is S or O.

Further examples of compounds include structures according to formula(IVa) to (IVd)

wherein mCat+ or mAn− is an organic or inorganic positively/negativelycharged counterion and

m is an integer 0-3;

q is an integer 1-5;

alk is a chain of 1-5 carbon atoms optionally containing one or moredouble or triple bonds;

Y is S, O or CH₂;

Z is OH;

n is an integer 0-3;

X is OH or O⁻ or an amide or ester conjugate thereof;

Ra₁ is H, SO₃ ⁻, sulfonamide, halogen, or a further ring fused to anadjacent carbon atom;

Rc₁ is alkyl or substituted alkyl; and

Rd is H, alkyl, substituted alkyl, aryl, substituted aryl, halogen,carboxy, sulphonamide, or sulfonic acid.

Further examples of compounds include structures according to formula(Va) to (Vd):

wherein mCat+ or mAn− is an organic or inorganic positively/negativelycharged counterion and

m is an integer 0-3;

q is an integer 1-5;

alk is a chain of 1-5 carbon atoms optionally containing one or moredouble or triple bonds;

Y is S, O or CH₂;

Z is OH;

n is an integer 0-3; and

X is OH or O⁻ or an amide or ester conjugate thereof.

Further examples of compounds include structures according to formula(VIa) to (VId):

wherein mCat+ or mAn− is an organic or inorganic positively/negativelycharged counterion and

m is an integer 0-3;

q is an integer 1-5;

alk is a chain of 1-5 carbon atoms optionally containing one or moredouble or triple bonds;

t is an integer 1-6;

Y is S, O or CH₂;

Z is OH;

n is an integer 0-3; and

X is OH or O⁻ or an amide or ester conjugate thereof.

alk is an alkyl, alkenyl or alkynyl chain of 1-5 carbon atoms optionallycontaining one or more double or triple bonds. Alk can be a group (CH₂)rwhere r is 1-5. Alk can be (CH₂)₃. Alternatively the carbon chain maycontain one or more double bonds or triple bonds. The chain may containa linkage —CH₂—CH═CH—CH₂—, optionally with further CH₂ groups.

The chain may contain a linkage —CH₂—C≡C—CH₂—, optionally with furtherCH₂ groups.

In any of the examples given in formula VII to VIII; r can equal 3.

In any of the examples given in formula I to VIII; q can equal 5.

In any of the examples given in formula III, formula VI or formula VII;t can equal 4.

In any of the examples given in formula I to VI; n can equal 1-3. In anyof the examples given in formula I to VI; n can equal 1. In any of theexamples given in formula I to VI; n can be an integer 0-1. Where n is1, the OH group can be at any position on the ring. The OH group can beat the 4 position. Where n is 2 or 3, the OH groups can be at anypositions on the phenyl ring.

In any of the examples given in formula I to VI; when n is zero, Y canequal O or S and not CH₂.

In any of the examples given in formula I to VI; Y can equal O.

In any of the examples given in formula I to VI; Y can equal O. Where Yis O, n can be 0-3. Where Y is CH₂, n can be 1-3.

Further examples of compounds include structures according to formula(VIIa) to (VIId):

wherein mCat+ or mAn− is an organic or inorganic positively/negativelycharged counterion and

m is an integer 0-3;

q is an integer 1-5;

r is an integer 1-5;

t is an integer 1-6; and

X is OH or O⁻ or an amide or ester conjugate thereof.

Further examples of compounds include structures according to formula(VIIIa) to (VIIId):

wherein mCat+ or mAn− is an organic or inorganic positively/negativelycharged counterion and

m is an integer 0-3;

q is an integer 1-5;

r is an integer 1-5; and

X is OH or O⁻ or an amide or ester conjugate thereof.

A particularly useful compound is a nucleotide or oligonucleotidelabelled with a dye as described herein.

The labelled nucleotide or oligonucleotide may have the label attachedto the nitrogen atom of indole via an alkyl-carboxy group to form anamide. The labelled nucleotide or oligonucleotide may have the labelattached to the C5 position of a pyrimidine base or the C7 position of a7-deaza purine base through a linker moiety.

The labelled nucleotide or oligonucleotide may also have a blockinggroup covalently attached to the ribose or deoxyribose sugar of thenucleotide. The blocking group may be attached at any position on theribose or deoxyribose sugar. In particular embodiments, the blockinggroup is at the 3′ OH position of the ribose or deoxyribose sugar of thenucleotide.

Provided herein are kits including two or more nucleotides wherein atleast one nucleotide is a nucleotide labelled with a compound of thepresent disclosure. The kit may include two or more labellednucleotides. The nucleotides may be labelled with two or morefluorescent labels. Two or more of the labels may be excited using asingle excitation source, which may be a laser. For example, theexcitation bands for the two or more labels may be at least partiallyoverlapping such that excitation in the overlap region of the spectrumcauses both labels to emit fluorescence. In particular embodiments, theemission from the two or more labels will occur in different regions ofthe spectrum such that presence of at least one of the labels can bedetermined by optically distinguishing the emission.

The kit may contain four labelled nucleotides, where the first of fournucleotides is labelled with a compound as disclosed herein. In such akit, the second, third, and fourth nucleotides can each be labelled witha compound that is optionally different from the label on the firstnucleotide and optionally different from the labels on each other. Thus,one or more of the compounds can have a distinct absorbance maximumand/or emission maximum such that the compound(s) is(are)distinguishable from other compounds. For example, each compound canhave a distinct absorbance maximum and/or emission maximum such thateach of the compounds is distinguishable from the other three compounds.It will be understood that parts of the absorbance spectrum and/oremission spectrum other than the maxima can differ and these differencescan be exploited to distinguish the compounds. The kit may be such thattwo or more of the compounds have a distinct absorbance maximum above600 nm. The compounds of the invention typically absorb light in theregion above 640 nm.

The compounds, nucleotides or kits that are set forth herein may be usedto detect, measure or identify a biological system (including, forexample, processes or components thereof). Exemplary techniques that canemploy the compounds, nucleotides or kits include sequencing, expressionanalysis, hybridisation analysis, genetic analysis, RNA analysis,cellular assay (e.g. cell binding or cell function analysis), or proteinassay (e.g. protein binding assay or protein activity assay). The usemay be on an automated instrument for carrying out a particulartechnique, such as an automated sequencing instrument. The sequencinginstrument may contain two lasers operating at different wavelengths.

Disclosed herein is a method of synthesising compounds of thedisclosure. A compound of formula (X) and/or (X1), (X2) (X3) or (X4) ora salt thereof may be used as a starting material for the synthesis ofsymmetrical or unsymmetrical polymethine dyes:

or a salt thereof wherein Ra₁ is H, SO₃ ⁻, sulfonamide, halogen, or afurther ring fused to an adjacent carbon atom; Rc₁ is alkyl orsubstituted alkyl; Ar is an aromatic group and R is an alkyl group.Where specific examples of 4-hydroxyphenyl are shown, further hydroxylgroups may also be substituted on the ring in cases where n is greaterthan one. r can be equal to 3.

Disclosed herein is a method of synthesising compounds of thedisclosure. A compound of formula (X5) or a salt thereof may be used asa starting material for the synthesis of symmetrical or unsymmetricalpolymethine dyes:

As used herein, the term “alkyl” refers to C₁-C₂₀ hydrocarbon and mayinclude C₃-C₁₀ non-aromatic carbocyclic rings. In particular embodimentsthe alkyl groups are C₁-C₆ alkyl which refers to saturated, straight- orbranched-chain hydrocarbon radicals containing between one and sixcarbon atoms, respectively. In particular cases, alkyl groups mayinclude one or more unsaturated groups, and thus include alkenyl andalkynyl.

The term “halogen” as used herein refers to fluoro-(hereafter designatedas F), chloro-(hereafter designated as Cl), bromo-(hereafter designatedas Br) or iodo-(hereafter designated as I), and usually relates tosubstitution for a hydrogen atom in an organic compound, thissubstitution is optionally a full substitution for the hydrogen.

The term “substituted alkyl”, refers to alkyl, alkenyl or alkynyl groupsas defined above where they may optionally be further substituted with,but not limited to, halo, cyano, SO₃ ⁻, SRa, ORa, NRbRc, oxo, CONRbRc,COOH and COORb. Ra, Rb and Rc may be each independently selected from H,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, aryl and substituted aryl. Further, saidsubstituted alkyl, substituted alkenyl and substituted alkynyl mayoptionally be interrupted by at least one hetero atom or group selectedfrom O, NRb, S(O)_(t) where t is 0 to 2, and the like. Substituted alkylalso covers group such as benzyl where the alkyl groups is comprises afurther aryl or substituted aryl moiety.

Dyes according to the present disclosure may be synthesised from avariety of different starting materials, includingN-propyl-4-hydroxyphenylether substituted indoles. The dyes may be madesymmetrically, such that the same indole is at both end of thepolymethine chain, or unsymmetrically such that different indoles are ateither end of the chromophore. Methods for preparing polymethine dyesare well known in the art.

According to an aspect of the disclosure there are provided dyecompounds suitable for attachment to substrate moieties, particularlycomprising linker groups to enable attachment to substrate moieties.Substrate moieties can be virtually any molecule or substance to whichthe dyes of the disclosure can be conjugated and, by way of non-limitingexample, may include nucleosides, nucleotides, polynucleotides,carbohydrates, ligands, particles, solid surfaces, organic and inorganicpolymers, chromosomes, nuclei, living cells and combinations orassemblages thereof. The dyes can be conjugated by an optional linker bya variety of means including hydrophobic attraction, ionic attractionand covalent attachment. Particularly the dyes are conjugated to thesubstrate by covalent attachment. More particularly the covalentattachment is by means of a linker group.

The conjugation of the dye compound to the substrate can be carried outvia carboxyl group COX, which can be turned into an amide or ester.

The dyes according to the present disclosure may include a reactivelinker group at one of the substituent positions for covalent attachmentof the dye to another molecule. Reactive linking groups are moietiescapable of forming a bond (e.g. a covalent or non-covalent bond). In aparticular embodiment the linker may be a cleavable linker. Use of theterm “cleavable linker” is not meant to imply that the whole linker isrequired to be removed. The cleavage site can be located at a positionon the linker that results in part of the linker remaining attached tothe dye and/or substrate moiety after cleavage. Cleavable linkers maybe, by way of non-limiting example, electrophilically cleavable linkers,enzymatically cleavable linkers, nucleophilically cleavable linkers,photocleavable linkers, cleavable under reductive conditions (forexample disulfide or azide containing linkers), oxidative conditions,cleavable via use of safety-catch linkers and cleavable by eliminationmechanisms. The use of a cleavable linker to attach the dye compound toa substrate moiety provides the option of removing the label, forexample after detection, thereby avoiding any interfering signal indownstream steps.

Useful linker groups may be found in PCT publication numberWO2004/018493 (herein incorporated by reference) examples of whichinclude linkers that, may be cleaved using water-soluble phosphines orwater-soluble transition metal catalysts formed from a transition metaland at least partially water-soluble ligands. In aqueous solution thelatter form at least partially water-soluble transition metal complexes.Such cleavable linkers can be used to connect bases of nucleotides tolabels such as the dyes set forth herein.

Particular linkers may be found in PCT publication number WO2004/018493(herein incorporated by reference) such as those that include moietiesof the formula:

(wherein X is selected from the group comprising O, S, NH and NQ whereinQ is a C1-10 substituted or unsubstituted alkyl group, Y is selectedfrom the group comprising O, S, NH and N(allyl), T is hydrogen or aC₁-C₁₀ substituted or unsubstituted alkyl group and * indicates wherethe moiety is connected to the remainder of the nucleotide ornucleoside).

In particular embodiments, the length of the linker between afluorescent dye (fluorophore) and a guanine base can be altered, forexample, by introducing a polyethylene glycol spacer group, therebyincreasing the fluorescence intensity compared to the same fluorophoreattached to the guanine base through other linkages known in the art.Exemplary linkers and their properties are set forth in GB patentapplication number 0517097.2, published as WO07020457, (hereinincorporated by reference). The design of linkers, and especially theirincreased length, can allow improvements in the brightness offluorophores attached to the guanine bases of guanosine nucleotides whenincorporated into polynucleotides such as DNA. Thus, when the dye is foruse in any method of analysis which employs detection of a fluorescentdye label attached to a guanine-containing nucleotide, it can beadvantageous to use a linker having a spacer group of formula—((CH₂)₂O)_(n)— wherein n is an integer between 2 and 50, for example,as described in WO07020457.

The present disclosure further provides conjugates of nucleosides andnucleotides labelled with one or more of the dyes set forth herein(modified nucleotides). Labelled nucleosides and nucleotides are usefulfor labelling polynucleotides formed by enzymatic synthesis, such as, byway of non-limiting example, in PCR amplification, isothermalamplification, solid phase amplification, polynucleotide sequencing(e.g. solid phase sequencing), nick translation reactions and the like.

Nucleosides and nucleotides may be labelled at sites on the sugar ornucleobase. As known in the art, a “nucleotide” consists of anitrogenous base, a sugar, and one or more phosphate groups. In RNA thesugar is ribose and in DNA is a deoxyribose, i.e. a sugar lacking ahydroxyl group that is present in ribose. The nitrogenous base is aderivative of purine or pyrimidine. The purines can be adenine (A) orguanine (G), and the pyrimidines can be cytosine (C), thymine (T) or inthe context of RNA, uracil (U). The C-1 atom of deoxyribose is bonded toN-1 of a pyrimidine or N-9 of a purine. A nucleotide is also a phosphateester of a nucleoside, with esterification occurring on the hydroxylgroup attached to the C-3 or C-5 of the sugar. Nucleotides are usuallymono, di- or triphosphates.

A “nucleoside” is structurally similar to a nucleotide but is missingthe phosphate moieties. An example of a nucleoside analog would be onein which the label is linked to the base and there is no phosphate groupattached to the sugar molecule.

Although the base is usually referred to as a purine or pyrimidine, theskilled person will appreciate that derivatives and analogues areavailable which do not alter the capability of the nucleotide ornucleoside to undergo Watson-Crick base pairing. “Derivative” or“analogue” means a compound or molecule whose core structure is the sameas, or closely resembles that of a parent compound but which has achemical or physical modification, such as, for example, a different oradditional side group, which allows the derivative nucleotide ornucleoside to be linked to another molecule. For example, the base maybe a deazapurine. In particular embodiments, the derivatives are capableof undergoing Watson-Crick pairing. “Derivative” and “analogue” alsoinclude, for example, a synthetic nucleotide or nucleoside derivativehaving modified base moieties and/or modified sugar moieties. Suchderivatives and analogues are discussed in, for example, Scheit,Nucleotide analogs (John Wiley & Son, 1980) and Uhlman et al., ChemicalReviews 90:543-584, 1990. Nucleotide analogues can also have modifiedphosphodiester linkages including phosphorothioate, phosphorodithioate,alkyl-phosphonate, phosphoranilidate, phosphoramidate linkages and thelike.

A dye may be attached to any position on a nucleotide base, for example,through a linker. In particular embodiments Watson-Crick base pairingcan still be carried out for the resulting analogue. Particularnucleobase labelling sites include the C5 position of a pyrimidine baseor the C7 position of a 7-deaza purine base. As described above a linkergroup may be used to covalently attach a dye to the nucleoside ornucleotide.

In particular embodiments the labelled nucleoside or nucleotide may beenzymatically incorporable and enzymatically extendable. Accordingly alinker moiety may be of sufficient length to connect the nucleotide tothe compound such that the compound does not significantly interferewith the overall binding and recognition of the nucleotide by a nucleicacid replication enzyme. Thus, the linker can also comprise a spacerunit. The spacer distances, for example, the nucleotide base from acleavage site or label.

Nucleosides or nucleotides labelled with dyes of the disclosure may havethe formula:

Where Dye is a dye compound according to the present disclosure, B is anucleobase, such as, for example uracil, thymine, cytosine, adenine,guanine and the like and L is an optional linker group which may or maynot be present. R′ can be H, monophosphate, diphosphate, triphosphate,thiophosphate, a phosphate ester analog, —O— attached to a reactivephosphorous containing group or —O— protected by a blocking group. R″can be H, OH, a phosphoramidite or a 3′OH blocking group and R′″ is H orOH.

Where R″ is phosphoramidite, R′ is an acid-cleavable hydroxyl protectinggroup which allows subsequent monomer coupling under automated synthesisconditions.

In a particular embodiment the blocking group is separate andindependent of the dye compound, i.e. not directly attached to it. In analternative embodiment the dye may comprise all or part of the 3′OHblocking group. Thus R″ can be a 3′OH blocking group which may or maynot comprise a dye compound disclosed herein.

In still yet another alternative embodiment there is no blocking groupon the 3′ carbon of the pentose sugar and the dye (or dye and linkerconstruct) attached to the base, for example, can be of a size orstructure sufficient to act as a block to the incorporation of a furthernucleotide. Thus the block can be due to steric hindrance or can be dueto a combination of size, charge and structure, whether or not the dyeis attached to the 3′ position of the sugar.

In still yet another alternative embodiment the blocking group ispresent on the 2′ or 4′ carbon of the pentose sugar and can be of a sizeor structure sufficient to act as a block to the incorporation of afurther nucleotide. The use of a blocking group allows polymerisation tobe controlled, such as by stopping extension when a modified nucleotideis incorporated. If the blocking effect is reversible, for example byway of non-limiting example by changing chemical conditions or byremoval of a chemical block, extension can be stopped at certain pointsand then allowed to continue.

In another particular embodiment a 3′OH blocking group will comprisemoieties disclosed in WO2004/018497 (herein incorporated by reference).For example the blocking group may be azidomethyl (CH₂N₃) or allyl.

In a particular embodiment a linker (between dye and nucleotide) and ablocking group are both present and are separate moieties. In particularembodiments the linker and blocking group are both cleavable undersubstantially similar conditions. Thus deprotection and deblockingprocesses may be more efficient since only a single treatment will berequired to remove both the dye compound and the block. However, in someembodiments a linker and blocking group need not be cleavable undersimilar conditions, instead being individually cleavable under distinctconditions.

This disclosure also encompasses polynucleotides incorporating dyecompounds. Such polynucleotides may be DNA or RNA comprised respectivelyof deoxyribonucleotides or ribonucleotides joined in phosphodiesterlinkage. Polynucleotides according to the disclosure may comprisenaturally occurring nucleotides, non-naturally occurring (or modified)nucleotides other than the modified nucleotides of the disclosure or anycombination thereof, in combination with at least one modifiednucleotide (e.g. labelled with a dye compound) set forth herein.Polynucleotides according to the disclosure may also include non-naturalbackbone linkages and/or non-nucleotide chemical modifications. Chimericstructures comprised of mixtures of ribonucleotides anddeoxyribonucleotides comprising at least one modified nucleotideaccording to the disclosure are also contemplated.

Modified nucleotides (or nucleosides) comprising a dye compoundaccording to the present disclosure may be used in any method ofanalysis such as methods that include detection of a fluorescent labelattached to a nucleotide or nucleoside, whether on its own orincorporated into or associated with a larger molecular structure orconjugate. In this context the term “incorporated into a polynucleotide”can mean that the 5′ phosphate is joined in phosphodiester linkage tothe 3′ hydroxyl group of a second (modified or unmodified) nucleotide,which may itself form part of a longer polynucleotide chain. The 3′ endof a modified nucleotide set forth herein may or may not be joined inphosphodiester linkage to the 5′ phosphate of a further (modified orunmodified) nucleotide. Thus, in one non-limiting embodiment thedisclosure provides a method of detecting a modified nucleotideincorporated into a polynucleotide which comprises: (a) incorporating atleast one modified nucleotide of the disclosure into a polynucleotideand (b) detecting the modified nucleotide(s) incorporated into thepolynucleotide by detecting the fluorescent signal from the dye compoundattached to said modified nucleotide(s).

This method can include: a synthetic step (a) in which one or moremodified nucleotides according to the disclosure are incorporated into apolynucleotide and a detection step (b) in which one or more modifiednucleotide(s) incorporated into the polynucleotide are detected bydetecting or quantitatively measuring their fluorescence.

In one embodiment of the present disclosure at least one modifiednucleotide is incorporated into a polynucleotide in a synthetic step bythe action of a polymerase enzyme. However, other methods of joiningmodified nucleotides to polynucleotides, such as for example chemicaloligonucleotide synthesis or ligation of labelled oligonucleotides tounlabelled oligonucleotides can be used. Therefore, the term“incorporating”, when used in reference to a nucleotide andpolynucleotide, can encompass polynucleotide synthesis by chemicalmethods as well as enzymatic methods.

In a specific embodiment a synthetic step is carried out and mayoptionally comprise incubating a template polynucleotide strand with areaction mixture comprising fluorescently labelled modified nucleotidesof the disclosure. A polymerase can also be provided under conditionswhich permit formation of a phosphodiester linkage between a free 3′hydroxyl group on a polynucleotide strand annealed to the templatepolynucleotide strand and a 5′ phosphate group on the modifiednucleotide. Thus, a synthetic step can include formation of apolynucleotide strand as directed by complementary base-pairing ofnucleotides to a template strand.

In all embodiments of the method, the detection step may be carried outwhilst the polynucleotide strand into which the modified nucleotides areincorporated is annealed to a template strand, or after a denaturationstep in which the two strands are separated. Further steps, for examplechemical or enzymatic reaction steps or purification steps, may beincluded between a synthetic step and a detection step. In particular,the target strand incorporating the modified nucleotide(s) may beisolated or purified and then processed further or used in a subsequentanalysis. By way of example, target polynucleotides labelled withmodified nucleotide(s) in a synthetic step may be subsequently used aslabelled probes or primers. In other embodiments the product of asynthetic step set forth herein may be subject to further reaction stepsand, if desired, the product of these subsequent steps can be purifiedor isolated.

Suitable conditions for a synthetic step will be well known to thosefamiliar with standard molecular biology techniques. In one embodiment asynthetic step may be analogous to a standard primer extension reactionusing nucleotide precursors, including modified nucleotides set forthherein, to form an extended target strand complementary to the templatestrand in the presence of a suitable polymerase enzyme. In otherembodiments a synthetic step may itself form part of an amplificationreaction producing a labelled double stranded amplification productcomprised of annealed complementary strands derived from copying oftarget and template polynucleotide strands.

Other exemplary synthetic steps include nick translation, stranddisplacement polymerisation, random primed DNA labelling etc. Aparticularly useful polymerase enzyme for a synthetic step is one thatis capable of catalysing the incorporation of one or more of themodified nucleotides set forth herein. A variety of naturally occurringor modified polymerases can be used. By way of example, a thermostablepolymerase can be used for a synthetic reaction that is carried outusing thermocycling conditions, whereas a thermostable polymerase maynot be desired for isothermal primer extension reactions. Suitablethermostable polymerases which are capable of incorporating the modifiednucleotides according to the disclosure include those described in WO2005/024010 or WO06120433, each of which is incorporated herein byreference. In synthetic reactions which are carried out at lowertemperatures such as 37° C., polymerase enzymes need not necessarily bethermostable polymerases, therefore the choice of polymerase will dependon a number of factors such as reaction temperature, pH,strand-displacing activity and the like.

In specific non-limiting embodiments the disclosure encompasses methodsof nucleic acid sequencing, re-sequencing, whole genome sequencing,single nucleotide polymorphism scoring, or any other applicationinvolving the detection of the modified nucleotide or nucleosidelabelled with dyes set forth herein when incorporated into apolynucleotide. Any of a variety of other applications benefitting fromthe use of polynucleotides labelled with the modified nucleotidescomprising fluorescent dyes can use modified nucleotides or nucleosideslabelled with dyes set forth herein.

In a particular embodiment the disclosure provides use of modifiednucleotides comprising dye compounds according to the disclosure in apolynucleotide sequencing-by-synthesis reaction. Sequencing-by-synthesisgenerally involves sequential addition of one or more nucleotides oroligonucleotides to a growing polynucleotide chain in the 5′ to 3′direction using a polymerase or ligase in order to form an extendedpolynucleotide chain complementary to the template nucleic acid to besequenced. The identity of the base present in one or more of the addednucleotide(s) can be determined in a detection or “imaging” step. Theidentity of the added base may be determined after each nucleotideincorporation step. The sequence of the template may then be inferredusing conventional Watson-Crick base-pairing rules. The use of themodified nucleotides labelled with dyes set forth herein fordetermination of the identity of a single base may be useful, forexample, in the scoring of single nucleotide polymorphisms, and suchsingle base extension reactions are within the scope of this disclosure.

In an embodiment of the present disclosure, the sequence of a templatepolynucleotide is determined by detecting the incorporation of one ormore nucleotides into a nascent strand complementary to the templatepolynucleotide to be sequenced through the detection of fluorescentlabel(s) attached to the incorporated nucleotide(s). Sequencing of thetemplate polynucleotide can be primed with a suitable primer (orprepared as a hairpin construct which will contain the primer as part ofthe hairpin), and the nascent chain is extended in a stepwise manner byaddition of nucleotides to the 3′ end of the primer in apolymerase-catalysed reaction.

In particular embodiments each of the different nucleotide triphosphates(A, T, G and C) may be labelled with a unique fluorophore and alsocomprises a blocking group at the 3′ position to prevent uncontrolledpolymerisation. Alternatively one of the four nucleotides may beunlabelled (dark). The polymerase enzyme incorporates a nucleotide intothe nascent chain complementary to the template polynucleotide, and theblocking group prevents further incorporation of nucleotides. Anyunincorporated nucleotides can be washed away and the fluorescent signalfrom each incorporated nucleotide can be “read” optically by suitablemeans, such as a charge-coupled device using laser excitation andsuitable emission filters. The 3′-blocking group and fluorescent dyecompounds can then be removed (deprotected), (simultaneously orsequentially) to expose the nascent chain for further nucleotideincorporation. Typically the identity of the incorporated nucleotidewill be determined after each incorporation step but this is notstrictly essential. Similarly, U.S. Pat. No. 5,302,509 (which isincorporated herein by reference) discloses a method to sequencepolynucleotides immobilised on a solid support.

The method, as exemplified above, utilizes the incorporation offluorescently labelled, 3′-blocked nucleotides A, G, C and T into agrowing strand complementary to the immobilised polynucleotide, in thepresence of DNA polymerase. The polymerase incorporates a basecomplementary to the target polynucleotide, but is prevented fromfurther addition by the 3′-blocking group. The label of the incorporatednucleotide can then be determined and the blocking group removed bychemical cleavage to allow further polymerisation to occur. The nucleicacid template to be sequenced in a sequencing-by-synthesis reaction maybe any polynucleotide that it is desired to sequence. The nucleic acidtemplate for a sequencing reaction will typically comprise a doublestranded region having a free 3′ hydroxyl group which serves as a primeror initiation point for the addition of further nucleotides in thesequencing reaction. The region of the template to be sequenced willoverhang this free 3′ hydroxyl group on the complementary strand. Theoverhanging region of the template to be sequenced may be singlestranded but can be double-stranded, provided that a “nick is present”on the strand complementary to the template strand to be sequenced toprovide a free 3′ OH group for initiation of the sequencing reaction. Insuch embodiments sequencing may proceed by strand displacement. Incertain embodiments a primer bearing the free 3′ hydroxyl group may beadded as a separate component (e.g. a short oligonucleotide) whichhybridises to a single-stranded region of the template to be sequenced.Alternatively, the primer and the template strand to be sequenced mayeach form part of a partially self-complementary nucleic acid strandcapable of forming an intra-molecular duplex, such as for example ahairpin loop structure. Hairpin polynucleotides and methods by whichthey may be attached to solid supports are disclosed in Internationalapplication publication nos. WO0157248 and WO2005/047301, each of whichis incorporated herein by reference. Nucleotides can be addedsuccessively to a growing primer, resulting in synthesis of apolynucleotide chain in the 5′ to 3′ direction. The nature of the basewhich has been added may be determined, particularly but not necessarilyafter each nucleotide addition, thus providing sequence information forthe nucleic acid template. Thus, a nucleotide is incorporated into anucleic acid strand (or polynucleotide) by joining of the nucleotide tothe free 3′ hydroxyl group of the nucleic acid strand via formation of aphosphodiester linkage with the 5′ phosphate group of the nucleotide.

The nucleic acid template to be sequenced may be DNA or RNA, or even ahybrid molecule comprised of deoxynucleotides and ribonucleotides. Thenucleic acid template may comprise naturally occurring and/ornon-naturally occurring nucleotides and natural or non-natural backbonelinkages, provided that these do not prevent copying of the template inthe sequencing reaction.

In certain embodiments the nucleic acid template to be sequenced may beattached to a solid support via any suitable linkage method known in theart, for example via covalent attachment. In certain embodimentstemplate polynucleotides may be attached directly to a solid support(e.g. a silica-based support). However, in other embodiments of thedisclosure the surface of the solid support may be modified in some wayso as to allow either direct covalent attachment of templatepolynucleotides, or to immobilise the template polynucleotides through ahydrogel or polyelectrolyte multilayer, which may itself benon-covalently attached to the solid support.

Arrays in which polynucleotides have been directly attached tosilica-based supports are those for example disclosed in WO00006770(incorporated herein by reference), wherein polynucleotides areimmobilised on a glass support by reaction between a pendant epoxidegroup on the glass with an internal amino group on the polynucleotide.In addition, polynucleotides can be attached to a solid support byreaction of a sulphur-based nucleophile with the solid support, forexample, as described in WO2005/047301 (incorporated herein byreference). A still further example of solid-supported templatepolynucleotides is where the template polynucleotides are attached tohydrogel supported upon silica-based or other solid supports, forexample, as described in WO00/31148, WO01/01143, WO02/12566,WO03/014392, U.S. Pat. No. 6,465,178 and WO00/53812, each of which isincorporated herein by reference.

A particular surface to which template polynucleotides may beimmobilised is a polyacrylamide hydrogel. Polyacrylamide hydrogels aredescribed in the references cited above and in WO2005/065814, which isincorporated herein by reference.

DNA template molecules can be attached to beads or microparticles, forexample as described in U.S. Pat. No. 6,172,218 (which is incorporatedherein by reference). Attachment to beads or microparticles can beuseful for sequencing applications. Bead libraries can be prepared whereeach bead contains different DNA sequences. Exemplary libraries andmethods for their creation are described in Nature. 437, 376-380 (2005);Science. 309, 5741, 1728-1732 (2005), each of which is incorporatedherein by reference. Sequencing of arrays of such beads usingnucleotides set forth herein is within the scope of the disclosure.

Template(s) that are to be sequenced may form part of an “array” on asolid support, in which case the array may take any convenient form.Thus, the method of the disclosure is applicable to all types of highdensity arrays, including single-molecule arrays, clustered arrays andbead arrays. Modified nucleotides labelled with dye compounds of thepresent disclosure may be used for sequencing templates on essentiallyany type of array, including but not limited to those formed byimmobilisation of nucleic acid molecules on a solid support.

However, the modified nucleotides labelled with dye compounds of thedisclosure are particularly advantageous in the context of sequencing ofclustered arrays. In clustered arrays, distinct regions on the array(often referred to as sites, or features) comprise multiplepolynucleotide template molecules. Generally, the multiplepolynucleotide molecules are not individually resolvable by opticalmeans and are instead detected as an ensemble. Depending on how thearray is formed, each site on the array may comprise multiple copies ofone individual polynucleotide molecule (e.g. the site is homogenous fora particular single- or double-stranded nucleic acid species) or evenmultiple copies of a small number of different polynucleotide molecules(e.g. multiple copies of two different nucleic acid species). Clusteredarrays of nucleic acid molecules may be produced using techniquesgenerally known in the art. By way of example, WO 98/44151 andWO00/18957, each of which is incorporated herein, describe methods ofamplification of nucleic acids wherein both the template andamplification products remain immobilised on a solid support in order toform arrays comprised of clusters or “colonies” of immobilised nucleicacid molecules. The nucleic acid molecules present on the clusteredarrays prepared according to these methods are suitable templates forsequencing using the modified nucleotides labelled with dye compounds ofthe disclosure.

The modified nucleotides labelled with dye compounds of the presentdisclosure are also useful in sequencing of templates on single moleculearrays. The term “single molecule array” or “SMA” as used herein refersto a population of polynucleotide molecules, distributed (or arrayed)over a solid support, wherein the spacing of any individualpolynucleotide from all others of the population is such that it ispossible to individually resolve the individual polynucleotidemolecules. The target nucleic acid molecules immobilised onto thesurface of the solid support can thus be capable of being resolved byoptical means in some embodiments. This means that one or more distinctsignals, each representing one polynucleotide, will occur within theresolvable area of the particular imaging device used.

Single molecule detection may be achieved wherein the spacing betweenadjacent polynucleotide molecules on an array is at least 100 nm, moreparticularly at least 250 nm, still more particularly at least 300 nm,even more particularly at least 350 nm. Thus, each molecule isindividually resolvable and detectable as a single molecule fluorescentpoint, and fluorescence from said single molecule fluorescent point alsoexhibits single step photobleaching.

The terms “individually resolved” and “individual resolution” are usedherein to specify that, when visualised, it is possible to distinguishone molecule on the array from its neighbouring molecules. Separationbetween individual molecules on the array will be determined, in part,by the particular technique used to resolve the individual molecules.The general features of single molecule arrays will be understood byreference to published applications WO00/06770 and WO 01/57248, each ofwhich is incorporated herein by reference. Although one use of themodified nucleotides of the disclosure is in sequencing-by-synthesisreactions, the utility of the modified nucleotides is not limited tosuch methods. In fact, the nucleotides may be used advantageously in anysequencing methodology which requires detection of fluorescent labelsattached to nucleotides incorporated into a polynucleotide.

In particular, the modified nucleotides labelled with dye compounds ofthe disclosure may be used in automated fluorescent sequencingprotocols, particularly fluorescent dye-terminator cycle sequencingbased on the chain termination sequencing method of Sanger andco-workers. Such methods generally use enzymes and cycle sequencing toincorporate fluorescently labelled dideoxynucleotides in a primerextension sequencing reaction. So called Sanger sequencing methods, andrelated protocols (Sanger-type), utilize randomised chain terminationwith labelled dideoxynucleotides.

Thus, the present disclosure also encompasses modified nucleotideslabelled with dye compounds which are dideoxynucleotides lackinghydroxyl groups at both of the 3′ and 2′ positions, such modifieddideoxynucleotides being suitable for use in Sanger type sequencingmethods and the like.

Modified nucleotides labelled with dye compounds of the presentdisclosure incorporating 3′ blocking groups, it will be recognized, mayalso be of utility in Sanger methods and related protocols since thesame effect achieved by using modified dideoxy nucleotides may beachieved by using modified nucleotides having 3′-OH blocking groups:both prevent incorporation of subsequent nucleotides. Where nucleotidesaccording to the present disclosure, and having a 3′ blocking group areto be used in Sanger-type sequencing methods it will be appreciated thatthe dye compounds or detectable labels attached to the nucleotides neednot be connected via cleavable linkers, since in each instance where alabelled nucleotide of the disclosure is incorporated; no nucleotidesneed to be subsequently incorporated and thus the label need not beremoved from the nucleotide.

The present disclosure also provides kits including modified nucleosidesand/or nucleotides labelled with dyes. Such kits will generally includeat least one modified nucleotide or nucleoside labelled with a dye setforth herein together with at least one further component. The furthercomponent(s) may be one or more of the components identified in a methodset forth above or in the Examples section below. Some non-limitingexamples of components that can be combined into a kit of the presentdisclosure are set forth below.

In a particular embodiment, a kit can include at least one modifiednucleotide or nucleoside labelled with a dye set forth herein togetherwith modified or unmodified nucleotides or nucleosides. For example,modified nucleotides labelled with dyes according to the disclosure maybe supplied in combination with unlabelled or native nucleotides, and/orwith fluorescently labelled nucleotides or any combination thereof.Accordingly the kits may comprise modified nucleotides labelled withdyes according to the disclosure and modified nucleotides labelled withother, for example, prior art dye compounds. Combinations of nucleotidesmay be provided as separate individual components (e.g. one nucleotidetype per vessel or tube) or as nucleotide mixtures (e.g. two or morenucleotides mixed in the same vessel or tube).

Where kits comprise a plurality, particularly two, more particularlyfour, modified nucleotides labelled with a dye compound, the differentnucleotides may be labelled with different dye compounds, or one may bedark, with no dye compounds. Where the different nucleotides arelabelled with different dye compounds it is a feature of the kits thatsaid dye compounds are spectrally distinguishable fluorescent dyes. Asused herein, the term “spectrally distinguishable fluorescent dyes”refers to fluorescent dyes that emit fluorescent energy at wavelengthsthat can be distinguished by fluorescent detection equipment (forexample, a commercial capillary based DNA sequencing platform) when twoor more such dyes are present in one sample. When two modifiednucleotides labelled with fluorescent dye compounds are supplied in kitform, it is a feature of some embodiments that the spectrallydistinguishable fluorescent dyes can be excited at the same wavelength,such as, for example by the same laser. When four modified nucleotideslabelled with fluorescent dye compounds are supplied in kit form, it isa feature of some embodiments that two of the spectrally distinguishablefluorescent dyes can both be excited at one wavelength and the other twospectrally distinguishable dyes can both be excited at anotherwavelength. Particular excitation wavelengths are 532 nm, 630 nm to 700nm, particularly 660 nm.

In one embodiment a kit includes a modified nucleotide labelled with acompound of the present disclosure and a second modified nucleotidelabelled with a second dye wherein the dyes have a difference inabsorbance maximum of at least 10 nm, particularly 20 nm to 50 nm. Moreparticularly the two dye compounds have Stokes shifts of between 15-40nm where “Stokes shift” is the distance between the peak absorption andpeak emission wavelengths.

In a further embodiment a kit can further include two other modifiednucleotides labelled with fluorescent dyes wherein the dyes are excitedby the same laser at 488 nm to 550 nm, particularly 532 nm. The dyes canhave a difference in absorbance maximum of at least 10 nm, particularly20 nm to 50 nm. More particularly the two dye compounds can have Stokesshifts of between 20-40 nm. Still yet more particularly the two dyecompounds can have a different absorbance maximum below 640 nm,particularly below 600 nm. Particular dyes which are spectrallydistinguishable from polymethine dyes of the present disclosure andwhich meet the above criteria are polymethine analogues as described inU.S. Pat. No. 5,268,486 (for example Cy3) or WO 0226891 (Alexa 532;Molecular Probes A20106) or unsymmetrical polymethines as disclosed inU.S. Pat. No. 6,924,372, each of which is incorporated herein byreference. Alternative dyes include rhodamine analogues, for exampletetramethyl rhodamine and analagues thereof.

In an alternative embodiment, the kits of the disclosure may containnucleotides where the same base is labelled with two differentcompounds. A first nucleotide may be labelled with a compound of thedisclosure. A second nucleotide may be labelled with a spectrallydistinct compound, for example a ‘green’ dye absorbing at less than 600nm. A third nucleotide may be labelled as a mixture of the compound ofthe disclosure and the spectrally distinct compound, and the fourthnucleotide may be ‘dark’ and contain no label. In simple terms thereforethe nucleotides 1-4 may be labelled ‘green’, ‘red’, ‘red/green’, anddark.

To simplify the instrumentation further, four nucleotides can belabelled with a two dyes excited with a single laser, and thus thelabelling of nucleotides 1-4 may be ‘red 1’, ‘red 2’ ‘red 1/red 2’, anddark or ‘green 1’, ‘green 2’ ‘green 1/green 2’, and dark.

Nucleotides may contain two dyes of the present disclosure. Dyes whereRa₁ or Ra₂ is a further aromatic ring fused to adjacent carbons of theindole ring absorb at a longer wavelength than where the dyes do nothave the further aromatic conjugation. A kit may contain two or morenucleotides labelled with dyes of the disclosure. A kit may contain anucleotide labelled with a compound of the disclosure where each of Ra₁and Ra₂ is independently H, SO₃ ⁻, sulphonamide or halogen, and onenucleotide labelled with a compound of the disclosure where one or bothRa₁ and Ra₂ is a further ring fused to an adjacent carbon atom. Kits maycontain a further nucleotide where the nucleotide is labelled with a dyethat absorbs in the region of 520 nm to 560 nm. Kits may further containan unlabelled nucleotide.

Nucleotides may contain two dyes of the present disclosure. Dyes where pis one absorb at a shorter wavelength than where p is two. A kit maycontain two or more nucleotides labelled with dyes of the disclosure. Akit may contain a nucleotide labelled with a compound of the disclosurewhere p is one, and one nucleotide labelled with a compound of thedisclosure where p is two. Kits may contain a further nucleotide wherethe nucleotide is labelled with a third label. Kits may further containan unlabelled nucleotide or a nucleotide labelled with a fourth label.

Although kits are exemplified above in regard to configurations havingdifferent nucleotides that are labelled with different dye compounds, itwill be understood that kits can include 2, 3, 4 or more differentnucleotides that have the same dye compound.

In particular embodiments a kit may include a polymerase enzyme capableof catalyzing incorporation of the modified nucleotides into apolynucleotide. Other components to be included in such kits may includebuffers and the like. The modified nucleotides labelled with dyesaccording to the disclosure, and other any nucleotide componentsincluding mixtures of different nucleotides, may be provided in the kitin a concentrated form to be diluted prior to use. In such embodiments asuitable dilution buffer may also be included. Again, one or more of thecomponents identified in a method set forth herein can be included in akit of the present disclosure.

It is noted that, as used in this specification and the appended claims,the singular forms “a”, “an” and “the” include plural referents unlessexpressly and unequivocally limited to one referent. It will be apparentto those skilled in the art that various modifications and variationscan be made to various embodiments described herein without departingfrom the spirit or scope of the present teachings. Thus, it is intendedthat the various embodiments described herein cover other modificationsand variations within the scope of the appended claims and theirequivalents.

EXPERIMENTAL DETAILS Preparation of 4-(3-bromopropoxy)phenol

To a solution of hydroquinone (3 g, 27.2 mmol) in methanol (30 mL) wasadded under N₂ potassium hydroxide (2 g, 35.4 mmol) and1,3-dibromopropane (8.3 mL, 81.7 mmol). The reaction was stirred at 65°C. overnight. Then volatiles were removed under vacuum and diethyl ether(30 mL) was added. The organic layer was washed with an ammoniumchloride saturated aqueous solution and water (30 mL each). The organiclayer was dried with magnesium sulphate, filtered and volatiles wereremoved under vacuum. The resulting beige slurry was purified by flashchromatography on silica gel (eluent: petroleum ether/Ethyl Acetate70/30) providing 2.6 g of a white slurry. Yield: 41%. Proton NMR(CDCl₃): 6.87-6.73 (4H, m, H—Ar); 5.36 (1H, s, OH); 4.04 (2H, t, J=6.2Hz); 3.60 (2H, J=6.2 Hz); 2.28 (2H, q, J=6.2 Hz, H-2). 13C NMR (CDCl₃):152.8, 149.6, 116.1; 116.0; 66.7; 34.4; 30.2.

Preparation of 4-(2,3-dimethyl-3H-indol-3-yl)butane-1-sulfonic Acid

Phenylhydrazine (8.6 mmol, 850 μL) and 5-methyl-6-oxoheptanesulfonicacid (7.2 mmol, 1.5 g) in glacial acetic acid (19 ml) were stirred at130° C. for 3 hours. The reaction mixture was cooled down to roomtemperature and the solvent removed under vacuum. The residue waspurified by flash chromatography on silica gel (eluent:dichloromethane/methanol 80/20) providing 1.23 g of a pale yellow foam.Yield: 61%. MS (DUIS): E⁻: 280 (M−1); Proton NMR (D₂O): 7.42 (1H, d,J=7.8 Hz, H-4); 7.27 (1H, t, J=7.8 Hz, H-5); 7.24 (1H, d, J=7.8 Hz,H-7); 7.17 (1H, t, J=7.8 Hz, H-6); 2.59 (2H, dd, J=7.6, 8.9 Hz, H-11);2.21-2.13 (1H, m); 1.88-1.73 (2H, m, H-8); 1.52-1.43 (2H, m, H-10); 1.13(3H, s, CH₃ on C-3); 0.70-0.63 (1H, m, H-9a); 0.51-0.44 (1H, m, H-9b).¹³C NMR (D₂O): 190.8; 152.3; 143.7; 127.8; 125.7; 122.2; 118.6; 57.9;50.5; 48.9; 35.6; 24.0; 22.5; 21.8; 14.8.

Preparation of1-(3-(4-hydroxyphenoxy)propyl)-2,3-dimethyl-3-(4-sulfobutyl)-3H-indol-1-ium

4-(3-bromopropoxy)phenol (1.90 mmol, 438 mg) and of4-(2,3-dimethyl-3H-indol-3-yl)butane-1-sulfonic acid (1.58 mmol, 504 mg)in sulfolane (2 ml) were stirred at 120° C. for 4.5 h. The reactionmixture was cooled down to room temperature and was directly purified byflash chromatography on silica gel (eluent: dichloromethane/methanol65/35) providing 329 mg of a pale grey oil. Yield: 40%. MS (DUIS): E⁻:430 (M−1), E⁺: 432 (M+1); Proton NMR (TFA): 7.75-7.65 (4H, m,H-4/5/6/7); 6.98 (2H, d, J=8.9 Hz, Har on phenyl); 6.85 (2H, d, J=8.9Hz, Har on phenyl); 4.82 (2H, bt, J=6.6 Hz, H-12); 4.24-4.13 (2H, m,H-14); 3.69 (1H, s); 3.20-3.06 (2H, m, H-11); 2.91 (3H, s, CH₃ on C-2);2.63-2.52 (2H, m, H-13); 2.41-2.75 (2H, m, H-8); 1.90-1.83 (2H, m,H-10); 1.66 (3H, s, CH3 on C-3); 1.13-0.93 (2H, m, H-9).

Preparation of1-(3-(4-hydroxyphenoxy)propyl)-2,3,3-trimethyl-3H-indol-1-ium

4-(3-bromopropoxy)phenol (1.40 mmol, 324 mg) and trimethylindolenine(0.80 mmol, 129 μl) in sulfolane (1 ml) were stirred at 120° C. for 3hours. The reaction mixture was cooled down to room temperature and wasdirectly purified by flash chromatography on silica gel (eluent:dichloromethane/methanol 90/10) providing 260 mg of a pale grey oil.Yield: 84%. Proton NMR (MeOD): 7.06 (1H, d, J=7.2 Hz, H-4); 7.00 (1H,bt, J=7.8 Hz, H-5); 6.77-6.64 (5H, m, H-6 & H-on phenyl ortho & meta);6.58 (1H, d, J=7.8 Hz, H-7); 3.90 (2H, t, J=5.8 Hz, H-8), 3.71 (2H,J=6.5 Hz, H-10); 2.04 (2H, bq, J=6.1 Hz, H-9); 1.30 (6H, s, 2×CH3 onC-3). ¹³C NMR (MeOD): 162.7; 153.6; 152.3; 147.2; 138.6; 128.6; 122.7;119.5; 116.8; 116.6; 116.5; 103.3; 66.8; 45.1; 39.7; 30.6; 27.5.

Preparation of AF670POPO

Product Synthesis Scheme:

Dye Synthesis Procedure:

4-(2,3-dimethyl-3H-indol-3-yl)butane-1-sulfonic acid (204 mg, 400 μmol)and malonaldehyde Bis(phenylimine) monohydrochloride (114 mg, 440 μmol)were stirred in mixture of acetic anhydride/acetic acid (2/0.6 ml) at100° C. for 1.5 h. Potassium acetate (118 mg, 1.20 mmol) and thesulfonated benzindolium salt X (193 mg, 400 μmol) were added. Thereaction mixture was stirred at 100° C. for 3 h. Then it was cooled downto room temperature and diethyl ether (40 mL) was added. A dark bluesolid crashed out. It was filtered, washed with extra diethyl ether (40mL) and dissolved in water (25 mL) containing 30% of acetonitrile. Theresulting dark blue solution was filtered and purified by preparativeHPLC. The main blue coloured (absorption max 665 nm) fractions werecollected and solvents removed in vacuum. MS (DUIS): E⁻: 869 (M−1),434.1 (M−2/2).

Dyes DX-DX were prepared similarly using appropriate starting materials

In Table 1 spectral properties of some dyes prepared in this way(solutions in water, OD 0.2) compared with similar parameters of knownstructural analogue dye Std (Patent: P6240WO)

TABLE 1

Max. Max. Fluores- Abs. cence Dye R₁ R₂ (nm) (nm) Std Ph SO₂NH₂ 650 673(NR650C5) AF670POPO —(CH₂)₃—O—C₆H₄—pOH -Benz-SO₃ ⁻ 666 684 AF670POP—(CH₂)₃—O—C₆H₅ -Benz-SO₃ 665 684 AF670PPO —(CH₂)₃—C₆H₄—pOH -Benz-SO₃ 665683 AF670PPOM —(CH₂)₃—C₆H₄—pOMe -Benz-SO₃ 665 683 AF670PP —(CH₂)₃—C₆H₅-Benz-SO₃ 664 683

Preparation of AF550POPOS0

Product Synthesis Scheme:

Dye Synthesis Procedure:

1-(3-(4-hydroxyphenoxy)propyl)-2,3,3-trimethyl-3H-indol-1-ium (150 mg,385 μmol) and N,N′-diphenylformamidine (74 mg, 385 μmol) were stirred inacetic anhydride (2 mL) at 80° C. for 2 h. Acetic acid (0.5 mL),Potassium acetate (567 mg, 5.78 mmol) and the sulfonated indolium salt X(166 mg, 385 μmol) were added. The reaction mixture was stirred at 80°C. for 2.5 h. Then volatiles were removed under reduced pressure anddiethyl ether (20 mL) was added. A dark red solid crashed out. It wasfiltered, washed with extra diethyl ether (20 mL) and dissolved in water(15 mL) containing 5% of acetonitrile. The resulting dark red solutionwas filtered and purified by preparative HPLC. The main red coloured(absorption max 550 nm) fractions were collected and solvents removed invacuum. MS (DUIS): Non-acetylated E⁻: 671.0 (M−1); E⁺: 673.3 (M+1),774.4 (Et₃N⁺ salt).

In Table 2 spectral properties of some dyes prepared in this way(solutions in water, OD 0.2) compared with similar parameters of knownstructural analogue dyes (Patent: P6240WO)

TABLE 2

Max. Max. Fluores- Abs. cence Dye R₁ (nm) (nm) Std 1 Ph 550 576(NR550S0) Std 2 Me 545 559 (NR545S0) AF550POPOS0 —(CH₂)₃—O—C₆H₄—pOH 548563 AF550POPS0 —(CH₂)₃—O—C₆H₅ 547 566 AF550PPOS0 —(CH₂)₃—C₆H₄—pOH 548567 AF550PPS0 —(CH₂)₃—C₆H₅ 548 567

Dye AF670POPO Nucleotide Conjugate (FFA-AF670POPO)

Anhydrous DMA (3 mL), TSTU (18 mg, 58.8 μmol) and Hunig's Base (0.05 mL)were added to the dried sample of the dye AF670POPO (60 mg, 53.5 μmol).The blue colour of activated ester developed. The reaction mixture wasstirred at room temperature for 30 min. According to TLC (20% H2O inCH₃CN) the activation was completed. After activation was completed, thesolution of 5 pppA-LN3 as a triethylammonium salt (53 mg, 56.1 μmol) inDMA/water (0.5/0.3 mL) was added to the reaction. The reaction mixturewas stirred at room temperature under nitrogen atmosphere for 18 h. Thecoupling progress was checked by TLC (20% H2O in acetonitrile). Thereaction mixture was cooled down to ˜4° C. with an ice-bath, then a 10solution of 0.1 M TEAB (5 mL) in water was added and the mixture wasstirred at room temperature for 10 min. The reaction mixture was appliedto column with ˜25 g of DEAE Sephadex resin suspension in 0.05 M TEABsolution in water and washed with TEAB (concentration gradient from 0.1M up to 1 M). Coloured fractions were collected and evaporated thenco-evaporated again with water to remove more TEAB and vac down todryness. The residue was then re-dissolved in TEAB 0.1 M. This solutionwas filtered through a syringe filter 0.2 nm pore size into a corning 20flask. The product was purified by HPLC using C18 reverse phase columnwith acetonitrile-0.1 M TEAB. Yield 74% (based on optical density ofsolution using estimated extinction coefficient).

Dye AF550POPOS0 Nucleotide Conjugate (FFT-AF550POPOS0)

Anhydrous DMA (3 mL), TSTU (19 mg, 63.5 μmol) and Hunig's Base (0.05 mL)were added to the dried sample of the dye AF550POPOS0 (39 mg, 57.7μmol). The red colour of activated ester developed. The reaction mixturewas stirred at room temperature for 30 min. The solution of pppT-LN3 asa triethylammonium salt (59 mg, 63.5 μmol) in DMA/water (0.5/0.3 mL) wasadded to the reaction. The reaction mixture was stirred at roomtemperature under nitrogen atmosphere for 18 h. The coupling progresswas checked by TLC (20% H2O in acetonitrile). The reaction mixture wascooled down to ˜4° C. with an ice-bath, then a 10 solution of 0.1 M TEAB(5 mL) in water was added and the mixture was stirred at roomtemperature for 10 min. The reaction mixture was applied to column with˜25 g of DEAE Sephadex resin suspension in 0.05 M TEAB solution in waterand washed with TEAB (concentration gradient from 0.1 M up to 1 M).Coloured fractions were collected and evaporated then co-evaporatedagain with water to remove more TEAB and vac down to dryness. Theresidue was then re-dissolved in TEAB 0.1 M. This solution was filteredthrough a syringe filter 0.2 nm pore size into a corning 20 flask. Theproduct was purified by HPLC using C18 reverse phase column withacetonitrile-0.1 M TEAB. Yield 48% (based on optical density of solutionusing estimated extinction coefficient).

DESCRIPTION OF FIGURES

FIG. 1 FFAs labelled with AF670POPO and its analogues along withffA-NR650C5 were tested on modified Illumina HiseqX sequencing systems,in which increasing excitation power of both lasers (red and green) byca. 33% was deployed at cycle 101 & 251 for read 1 & 2 respectively (seesignals on FIG. 1).

FIGS. 2A and 2B Unexpectedly, in the case of ffA-NR650C5 (FIG. 2A), itwas observed that the red signals didn't respond proportionally to thepower change of red laser. Consequentially, the shape of the baseseparation scatterplots was altered at cycle 101. The top right circleis closer to the top left circle, meaning it is more difficult todistinguish between the respective labels. The occurring phenomenaindicated that NR650C5 was probably photo-saturated or near-saturated atthe maximum power density used on HiSeqX. This scatter plot changenegatively impacts sequencing quality. Interestingly, with AF670POPO andits analogues, a remarkable improvement was observed. Their signal wasincreased proportionally with laser power density change. The scatterplot maintained their separations and shape from cycle 100 to cycle 101when maximum power was deployed (FIG. 2B). The top right circle in FIG.2B is further from the top left circle than the top right circle in FIG.2A. Thus the labels are better separated in FIG. 2B.

FIG. 3 On the 550 nm analogues, it was interesting to observe that thepresence of hydroxyl on the phenyl ring has a detrimental impact on thefluorescence intensity (FIG. 3). The fluorescence of AF550POPS0 withoutthe phenolic OH was 3 times stronger than the AF550POPOS0 having the OH.

FIGS. 4A and 4B These new analogues offered opportunities to modulateand optimise scatterplot distribution for sequencing applications. Forexample, when T was labelled with AF550POPOS0 and A with a mixture ofAF670POPO and NR550S0, a nice square scatterplot was achieved (FIG. 4A).Furtherly replacing NR550S0 with AF550POPS0 for the green A showed anicely square scatterplot (FIG. 4B).

FIG. 5 Based on these new properties, ffT-LN3-AF550POPOS0 andffA-LN3-AF670POPO were employed to sequence a human template on amodified HiSeqX. A high quality 2×150 bp sequencing were achieved <1%error rate for read 1 and <1.5% error rate for read 2 (main sequencingmetrics shown on FIG. 5). The scatter plots obtained using the prior artlabels meant that a 2×150 bp read could not be completed.

The invention claimed is:
 1. A compound of the formula (I), or amesomeric form thereof:

wherein mCat+ or mAn− is an organic or inorganic positively/negativelycharged counterion; m is an integer 0-3; p is an integer 1 or 2; q is aninteger 1-5; alk is a chain of 1-5 carbon atoms optionally containingone or more double or triple bonds; Y is S or O; Z is OH; n is 0 or 1; Xis OH or O⁻, or an amide or ester conjugate thereof; each of Ra₁ and Ra₂is independently H, SO₃ ⁻, sulfonamide, halogen, or a further ring fusedto an adjacent carbon atom; and each of Rc₁ and Rc₂ is independentlyalkyl or substituted alkyl, wherein at least one of Ra₁ or Ra₂ is SO₃ ⁻,or Ra₁ or Ra₂ is a further ring fused to an adjacent carbon atom, thefurther ring having an SO₃ ⁻, or Rc₁ or Rc₂ is an alkyl sulfonic acidgroup.
 2. The compound according to claim 1, wherein (alk) is (CH₂)₃. 3.The compound according to claim 1, wherein q is
 5. 4. The compoundaccording to claim 1, wherein each of Ra₁ and Ra₂ is H or SO₃ ⁻.
 5. Thecompound according to claim 1, wherein Ra₁ or Ra₂ is a further ringfused to an adjacent carbon atom.
 6. The compound according to claim 1,wherein Rc₁ or Rc₂ is methyl, ethyl, propyl or —(CH₂)_(t)SO₃ ⁻ where tis 1-6.
 7. The compound according to claim 6, wherein either Rc₁ or Rc₂is —(CH₂)₄—SO₃ ⁻.
 8. The compound according to claim 1, wherein Y is O.9. The compound according claim 1, wherein n is
 1. 10. The compoundaccording to claim 1, wherein either indole moiety of formula (I) ispart of a structure:

wherein Rd is H, alkyl, substituted alkyl, aryl, substituted aryl,halogen, carboxy, sulfonamide, or sulfonic acid; and wherein Rc₁ isalkyl or substituted alkyl.
 11. The compound according to claim 1, whichis represented by formula (VIIIa), or a mesomeric form thereof:

wherein r is an integer 1-5.
 12. The compound according to claim 1,which is represented by formula (VIId), or a mesomeric form thereof:

wherein r is an integer 1-5; and t is an integer 1-6.
 13. A nucleotideor oligonucleotide labelled with a compound according to claim
 1. 14. Anucleotide or oligonucleotide labelled with a compound according toclaim 12, wherein the compound is attached to the nucleotide oroligonucleotide via an amide linkage formed from the C(═O)—X moiety. 15.The nucleotide or oligonucleotide according to claim 13, wherein thecompound is attached to the C5 position of a pyrimidine base or the C7position of a 7-deaza purine base of the nucleotide or oligonucleotidethrough a linker moiety.
 16. The nucleotide or oligonucleotide accordingto claim 13, further comprising a 3′ OH blocking group covalentlyattached to the ribose or deoxyribose sugar of the nucleotide oroligonucleotide.
 17. A kit comprising two or more nucleotides wherein atleast one nucleotide is a labelled nucleotide according to claim
 13. 18.The kit according to claim 17, wherein two of the labelled nucleotidesare excited using a single laser.
 19. The kit according to claim 18,wherein a first of four nucleotides is a labelled nucleotide accordingto claim 13, and the second, third, and fourth nucleotides are eachlabelled with a different compound, wherein each compound has a distinctabsorbance maximum and each of the compounds is distinguishable from theother three compounds.
 20. The kit according to claim 17, comprisingfour nucleotides wherein a first of the four nucleotides is a labellednucleotide according to claim 13, a second of the four nucleotides is alabelled nucleotide according to claim 13, a third nucleotide carries athird label and a fourth nucleotide is unlabelled (dark).
 21. The kitaccording to claim 17 comprising four nucleotides wherein a first of thefour nucleotides is a labelled nucleotide according to claim 13, asecond of the four nucleotides is a labelled nucleotide according toclaim 13, a third nucleotide carries a mixture of two labels and afourth nucleotide is unlabelled (dark).
 22. Use of a nucleotide oroligonucleotide according to claim 13 in sequencing, expressionanalysis, hybridisation analysis, genetic analysis, RNA analysis orprotein binding assays.
 23. Use according to claim 22, on an automatedsequencing instrument wherein said automated sequencing instrumentcomprises two lasers operating at different wavelengths.
 24. A method ofsynthesising a compound according to claim 1, utilising one of thefollowing starting materials:

or a salt thereof; wherein Ra₁ is H, SO₃ ⁻, sulfonamide, halogen, or afurther ring fused to an adjacent carbon atom; Rc₁ is alkyl orsubstituted alkyl; r is an integer 1-5; Ar is an aromatic group; and Ris an alkyl group.
 25. A method of synthesising a compound according toclaim 1, utilising the following starting material:


26. The compound according to claim 11, wherein X is OH and q is
 5. 27.The compound according to claim 12, wherein X is OH and q is
 5. 28. Thecompound according to claim 1, selected from the group consisting of:

and mesomeric forms thereof.
 29. The nucleotide or oligonucleotideaccording to claim 14, comprising the compound moiety:

or a mesomeric form thereof.
 30. A nucleotide or oligonucleotidelabelled with a compound according to claim 11, wherein the compound isattached to the nucleotide or oligonucleotide via an amide linkageformed from the C(═O)—X moiety.
 31. The nucleotide or oligonucleotideaccording to claim 30, comprising the compound moiety:

or a mesomeric form thereof.